Abstract

A significant proportion of high-grade serous ovarian carcinomas arise from the secretory cells of the fallopian tube. Exocytosis, the process through which cells release vesicles containing various growth factors and signaling molecules is important for cell-cell and cell-microenvironment communications. Importantly, defects in regulated-exocytosis have been shown to be involved in tumorigenesis. Understanding the molecular determinants of exocytosis in secretory cells has immense potential for developing new cancer biomarkers and therapeutic agents. Using HE4, a secreted glycoprotein and a biomarker of ovarian cancer as a surrogate marker in our in silico approach, we recently identified Tumor Necrosis Factor alpha-induced protein 2 (TNFAIP2; also known as B94) as a potential regulator of exocytosis. TNFAIP2 is a retinoic acid pathway target gene that is induced by tumor necrosis factor alpha (TNFa), interleukin 1-beta (IL1-beta), and lipopolysaccharides. TNFAIP2 is evolutionarily conserved, yet little is known about its biological function and its role in cancer. Its only structural domain is a Sec6 domain which is found in proteins of the exocyst complex and in those involved with cytokinesis.

Analysis of high-density tissue microarrays (TMAs) of all major cancer histotypes showed that TNFAIP2 is most highly expressed in ovarian serous carcinomas and certain lymphomas. Gene Ontology and Ingenuity Pathway Analyses of TNFAIP2 expression and co-expressed genes suggest a possible connection to tumors of the reproductive tract and a role in cell proliferation and migration. We interrogated the role of TNFAIP2 in these processes using RNA interference. Robust knock-down of TNFAIP2 expression using siRNA led to decrease in proliferation and migration of ovarian cancer cells. Analysis of sub-cellular localization shows that TNFAIP2 is localized to cell membrane and regions of the cytoplasm. To further understand the mechanism of TNFAIP2 function, we cloned full length TNFAIP2 and expressed it as a FLAG-StrepTactin tagged fusion protein, and the TNFAIP2 complex was tandem-affinity purified. The associated proteins were identified by mass spectrometry. TNFAIP2 interacts with various component of adherens junctions and vesicular trafficking, including E-cadherin, integrins, alpha, beta, and delta-catenins, and Rab proteins. Interestingly, TNFAIP2 also interacts with a group of receptor tyrosine kinases (RTKs), including AXL. Our results show that TNFAIP2 is an important mediator of endosome recycling and that it regulates the stability of certain receptor tyrosine kinases, including AXL and MER, by enhancing their processing through recycling-endosomes. Structure-function studying are ongoing as well as studies aimed at targeting TNFAIP2 in the setting of RTK activation.